Investigation of rpoS and dps Genes in Sodium Hypochlorite Resistance of Salmonella Enteritidis SE86 Isolated from Foodborne Illness Outbreaks in Southern Brazil

2012 ◽  
Vol 75 (3) ◽  
pp. 437-442 ◽  
Author(s):  
ANA CAROLINA RITTER ◽  
DONATELLA BACCIU ◽  
LUCÉLIA SANTI ◽  
WALTER ORLANDO BEYS da SILVA ◽  
MARILENE HENNING VAINSTEIN ◽  
...  

In Rio Grande do Sul, southern Brazil, Salmonella Enteritidis is one of the principal microorganisms responsible for foodborne disease. The present study was conducted to compare the sodium hypochlorite resistance of Salmonella Enteritidis SE86 with that of other strains of Salmonella Enteritidis isolated from different regions of the world and to investigate the involvement of the rpoS and dps genes in resistance to this disinfectant. We tested five Salmonella Enteritidis wild-type (WT) strains isolated from different countries, two mutant strains of Salmonella Enteritidis SE86, and two tagged (3XFLAG) strains of Salmonella Enteritidis SE86 for their resistance to sodium hypochlorite (200 ppm). The survival of the WT and attenuated strains was determined based on bacterial counts, and tagged proteins (Dps and RpoS) were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. None of the WT strains of Salmonella Enteritidis were totally inactivated after 20 min. The SE86 strain lacking dps was more sensitive to sodium hypochlorite than was the WT SE86 strain, with a 2-log reduction in counts after 1 min. The RpoS and Dps proteins were actively expressed under the conditions tested, indicating that in Salmonella Enteritidis SE86 these genes, which are expressed when in contact with sodium hypochlorite, are related to oxidative stress.

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.


2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.


1982 ◽  
Vol 152 (1) ◽  
pp. 166-174
Author(s):  
J A Mulder ◽  
G Venema

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.


1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.


1997 ◽  
Vol 9 (1) ◽  
pp. 32-38 ◽  
Author(s):  
Barbara C. Hegarty ◽  
Michael G. Levy ◽  
Robin F. Gager ◽  
Edward B. Breitschwerdt

Historically, considerable variation has been reported in the type and severity of clinical and hematologic abnormalities associated with canine ehrlichiosis. Because of difficulties associated with the isolation of intracellular monocytic Ehrlichia species in tissue culture systems, few E. canis isolates are available for comparative microbiologic studies. To address the issue of potential E. canis antigenic diversity in different regions of the world, dog sera reactive by indirect fluorescent antibody testing to E. canis (Florida) antigen were obtained from France, Israel, Italy, the United States, the Virgin Islands, and Zimbabwe. Ehrlichia canis proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and at least 5 sera from each region were stained by western immunoblotting. Antibody immunodominance was scored based upon staining intensity. There was relative homogeneity in the immunogenic protein reactions to E. canis antigens. Of the 58 E. canis reactive sera, 54 samples resulted in immunoblot patterns indicative of chronic ehrlichiosis. Four reactive sera (reciprocal titers of 160–2,560) did not recognize any genus-specific antigens resulting in protein bands between 22 and 29 kD, indicating serologic cross-reactivity with other microorganisms. Relatively homogenous immunoblot patterns, consistent with the reported immunoblot response of dogs with experimental chronic ehrlichiosis, were observed with sera from Arizona, France, Israel, North Carolina, Texas, and the Virgin Islands. In contrast, unique major proteins were observed in dog sera from Italy and Zimbabwe. Our results indicate that although relatively homogeneous, antigenic diversity may exist among E. canis organisms in different regions of the world.


1983 ◽  
Vol 217 (1208) ◽  
pp. 243-264 ◽  

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.


2000 ◽  
Vol 182 (23) ◽  
pp. 6698-6706 ◽  
Author(s):  
Chunhao Li ◽  
Linda Corum ◽  
David Morgan ◽  
Everett L. Rosey ◽  
Thaddeus B. Stanton ◽  
...  

ABSTRACT Spirochete periplasmic flagella (PFs), including those fromBrachyspira (Serpulina),Spirochaeta, Treponema, andLeptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructedflaA::cat,flaA::kan, andflaB1::kan mutants and newly constructed flaB2::cat andflaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found withflaA::cat,flaA::kan, andflaB1::kan mutants,flaB2::cat andflaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan,flaB2::cat, andflaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found thatflaA::cat andflaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.


1981 ◽  
Vol 200 (1) ◽  
pp. 83-91 ◽  
Author(s):  
I C Madley ◽  
B D Hames

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.


1988 ◽  
Vol 8 (3) ◽  
pp. 1011-1018 ◽  
Author(s):  
M K Sauer ◽  
D J Donoghue

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.


2003 ◽  
Vol 71 (8) ◽  
pp. 4351-4360 ◽  
Author(s):  
Vincent Idone ◽  
Stacy Brendtro ◽  
Robert Gillespie ◽  
Steve Kocaj ◽  
Erica Peterson ◽  
...  

ABSTRACT Streptococcus mutans is the principal acidogenic component of dental plaque that demineralizes tooth enamel, leading to dental decay. Cell-associated glucosyltransferases catalyze the sucrose-dependent synthesis of sticky glucan polymers that, together with glucan binding proteins, promote S. mutans adherence to teeth and cell aggregation. We generated an S. mutans Tn916 transposon mutant, GMS315, which is defective in sucrose-dependent adherence and significantly less cariogenic than the UA130 wild-type progenitor in germfree rats. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and N-terminal sequence analysis confirmed the absence of a 155-kDa glucosyltransferase S (Gtf-S) from GMS315 protein profiles. Mapping of the unique transposon insertion in GMS315 revealed disruption of a putative regulatory region located upstream of gcrR, a gene previously described by Sato et al. that shares significant amino acid identity with other bacterial response regulators (Y. Sato, Y. Yamamoto, and H. Kizaki, FEMS Microbiol. Lett. 186: 187-191, 2000). The gcrR regulator, which we call “tarC,” does not align with any of the 13 proposed two-component signal transduction systems derived from in silico analysis of the S. mutans genome, but rather represents one of several orphan response regulators in the genome. The results of Northern hybridization and/or real-time reverse transcription-PCR experiments reveal increased expression of both Gtf-S and glucan binding protein C (GbpC) in a tarC knockout mutant (GMS900), thereby supporting the notion that TarC acts as a negative transcriptional regulator. In addition, we noted that GMS900 has altered biofilm architecture relative to the wild type and is hypocariogenic in germfree rats. Taken collectively, these findings support a role for signal transduction in S. mutans sucrose-dependent adherence and aggregation and implicate TarC as a potential target for controlling S. mutans-induced cariogenesis.


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